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Alpha-scorpion toxin impairs a conformational change that leads to fast inactivation of muscle sodium channels.

Campos FV, Chanda B, Beirão PS, Bezanilla F - J. Gen. Physiol. (2008)

Bottom Line: We have used Ts3, an alpha-scorpion toxin from the Brazilian scorpion Tityus serrulatus, to analyze the effects of this family of toxins on the muscle sodium channels expressed in Xenopus oocytes.While the fluorescence-voltage (F-V) relationship of domain II was only slightly affected and the F-V of domain III remained unaffected in the presence of Ts3, the toxin significantly shifted the F-V of domain I to more positive potentials, which agrees with previous studies showing a strong coupling between domains I and IV.These results are consistent with the proposed model, in which Ts3 specifically impairs the fraction of the movement of the S4-DIV that allows fast inactivation to occur at normal rates.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637, USA.

ABSTRACT
Alpha-scorpion toxins bind in a voltage-dependent way to site 3 of the sodium channels, which is partially formed by the loop connecting S3 and S4 segments of domain IV, slowing down fast inactivation. We have used Ts3, an alpha-scorpion toxin from the Brazilian scorpion Tityus serrulatus, to analyze the effects of this family of toxins on the muscle sodium channels expressed in Xenopus oocytes. In the presence of Ts3 the total gating charge was reduced by 30% compared with control conditions. Ts3 accelerated the gating current kinetics, decreasing the contribution of the slow component to the ON gating current decay, indicating that S4-DIV was specifically inhibited by the toxin. In addition, Ts3 accelerated and decreased the fraction of charge in the slow component of the OFF gating current decay, which reflects an acceleration in the recovery from the fast inactivation. Site-specific fluorescence measurements indicate that Ts3 binding to the voltage-gated sodium channel eliminates one of the components of the fluorescent signal from S4-DIV. We also measured the fluorescent signals produced by the movement of the first three voltage sensors to test whether the bound Ts3 affects the movement of the other voltage sensors. While the fluorescence-voltage (F-V) relationship of domain II was only slightly affected and the F-V of domain III remained unaffected in the presence of Ts3, the toxin significantly shifted the F-V of domain I to more positive potentials, which agrees with previous studies showing a strong coupling between domains I and IV. These results are consistent with the proposed model, in which Ts3 specifically impairs the fraction of the movement of the S4-DIV that allows fast inactivation to occur at normal rates.

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Effects of Ts3 on the kinetic parameters of the ON gating currents. The parameters were obtained by fitting the ON gating current decay with function 1. Gating currents were recorded as described in Fig. 3 A. (A) Fast and slow time constants of the gating current decay. (B) Contribution of the slow component to the gating current decay. White circles show the slow component in control conditions and the black circles show the slow component in the presence of Ts3. Data shown as mean ± SEM, n = 4. *, existence of statistical difference (P < 0.05) between compared groups.
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fig4: Effects of Ts3 on the kinetic parameters of the ON gating currents. The parameters were obtained by fitting the ON gating current decay with function 1. Gating currents were recorded as described in Fig. 3 A. (A) Fast and slow time constants of the gating current decay. (B) Contribution of the slow component to the gating current decay. White circles show the slow component in control conditions and the black circles show the slow component in the presence of Ts3. Data shown as mean ± SEM, n = 4. *, existence of statistical difference (P < 0.05) between compared groups.

Mentions: It has been shown that the decay of the ON gating currents of sodium channels may be fitted to two components, and that the slow component is correlated with the movement of the voltage sensor of domain IV (Chanda and Bezanilla, 2002). The effects of Ts3 on the ON gating current kinetics were quantified by fitting the decay phase of the currents with a double exponential function (see Materials and methods). Fig. 4 A compares the voltage dependence of the fast and slow time constants of the gating current decay, obtained before and after the treatment with Ts3. From −90 until −30 mV the best fits were obtained with a double exponential function, but at more positive potentials, the two components merge into only one component. After the treatment with Ts3, the slow time constants became significantly faster while the fast time constant remained unaffected. Furthermore, Ts3 decreased the relative contribution of the slow component to the decay (Fig. 4 B). One should note that only at −50 and −60 mV the change in the relative contribution of the slow components was statistically significant. This could be due to the fact that at very hyperpolarized or depolarized potentials it is harder to distinguish the fast from the slow component. The specific effect of Ts3 on the slow component of the gating current decay supports the hypothesis that the bound toxin affects the movement of the S4 segment of domain IV.


Alpha-scorpion toxin impairs a conformational change that leads to fast inactivation of muscle sodium channels.

Campos FV, Chanda B, Beirão PS, Bezanilla F - J. Gen. Physiol. (2008)

Effects of Ts3 on the kinetic parameters of the ON gating currents. The parameters were obtained by fitting the ON gating current decay with function 1. Gating currents were recorded as described in Fig. 3 A. (A) Fast and slow time constants of the gating current decay. (B) Contribution of the slow component to the gating current decay. White circles show the slow component in control conditions and the black circles show the slow component in the presence of Ts3. Data shown as mean ± SEM, n = 4. *, existence of statistical difference (P < 0.05) between compared groups.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2483334&req=5

fig4: Effects of Ts3 on the kinetic parameters of the ON gating currents. The parameters were obtained by fitting the ON gating current decay with function 1. Gating currents were recorded as described in Fig. 3 A. (A) Fast and slow time constants of the gating current decay. (B) Contribution of the slow component to the gating current decay. White circles show the slow component in control conditions and the black circles show the slow component in the presence of Ts3. Data shown as mean ± SEM, n = 4. *, existence of statistical difference (P < 0.05) between compared groups.
Mentions: It has been shown that the decay of the ON gating currents of sodium channels may be fitted to two components, and that the slow component is correlated with the movement of the voltage sensor of domain IV (Chanda and Bezanilla, 2002). The effects of Ts3 on the ON gating current kinetics were quantified by fitting the decay phase of the currents with a double exponential function (see Materials and methods). Fig. 4 A compares the voltage dependence of the fast and slow time constants of the gating current decay, obtained before and after the treatment with Ts3. From −90 until −30 mV the best fits were obtained with a double exponential function, but at more positive potentials, the two components merge into only one component. After the treatment with Ts3, the slow time constants became significantly faster while the fast time constant remained unaffected. Furthermore, Ts3 decreased the relative contribution of the slow component to the decay (Fig. 4 B). One should note that only at −50 and −60 mV the change in the relative contribution of the slow components was statistically significant. This could be due to the fact that at very hyperpolarized or depolarized potentials it is harder to distinguish the fast from the slow component. The specific effect of Ts3 on the slow component of the gating current decay supports the hypothesis that the bound toxin affects the movement of the S4 segment of domain IV.

Bottom Line: We have used Ts3, an alpha-scorpion toxin from the Brazilian scorpion Tityus serrulatus, to analyze the effects of this family of toxins on the muscle sodium channels expressed in Xenopus oocytes.While the fluorescence-voltage (F-V) relationship of domain II was only slightly affected and the F-V of domain III remained unaffected in the presence of Ts3, the toxin significantly shifted the F-V of domain I to more positive potentials, which agrees with previous studies showing a strong coupling between domains I and IV.These results are consistent with the proposed model, in which Ts3 specifically impairs the fraction of the movement of the S4-DIV that allows fast inactivation to occur at normal rates.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637, USA.

ABSTRACT
Alpha-scorpion toxins bind in a voltage-dependent way to site 3 of the sodium channels, which is partially formed by the loop connecting S3 and S4 segments of domain IV, slowing down fast inactivation. We have used Ts3, an alpha-scorpion toxin from the Brazilian scorpion Tityus serrulatus, to analyze the effects of this family of toxins on the muscle sodium channels expressed in Xenopus oocytes. In the presence of Ts3 the total gating charge was reduced by 30% compared with control conditions. Ts3 accelerated the gating current kinetics, decreasing the contribution of the slow component to the ON gating current decay, indicating that S4-DIV was specifically inhibited by the toxin. In addition, Ts3 accelerated and decreased the fraction of charge in the slow component of the OFF gating current decay, which reflects an acceleration in the recovery from the fast inactivation. Site-specific fluorescence measurements indicate that Ts3 binding to the voltage-gated sodium channel eliminates one of the components of the fluorescent signal from S4-DIV. We also measured the fluorescent signals produced by the movement of the first three voltage sensors to test whether the bound Ts3 affects the movement of the other voltage sensors. While the fluorescence-voltage (F-V) relationship of domain II was only slightly affected and the F-V of domain III remained unaffected in the presence of Ts3, the toxin significantly shifted the F-V of domain I to more positive potentials, which agrees with previous studies showing a strong coupling between domains I and IV. These results are consistent with the proposed model, in which Ts3 specifically impairs the fraction of the movement of the S4-DIV that allows fast inactivation to occur at normal rates.

Show MeSH
Related in: MedlinePlus